RESUMEN
Human hepatocarcinogenesis is a complex process with many unresolved issues, including the cell of origin (differentiated and/or progenitor/stem cells) and the initial steps leading to tumor development. With the aim of providing new tools for studying hepatocellular carcinoma initiation and progression, we developed an innovative model based on primary human hepatocytes (PHHs) lentivirus-transduced with SV40LT+ST, HRASV12 with or without hTERT. The differentiation status of these transduced-PHHs was characterized by RNA sequencing (including lncRNAs), and the expression of some differentiation markers confirmed by RT-qPCR and immunofluorescence. In addition, their transformation capacity was assessed by colony formation in soft agar and tumorigenicity evaluated in immune-deficient mice. The co-expression of SV40LT+ST and HRASV12 in PHHs, in association or not with hTERT, led to the emergence of transformed clones. These clones exhibited a poorly differentiated cell phenotype with expression of stemness and mesenchymal-epithelial transition markers and gave rise to cancer stem cell subpopulations. In vivo, they resulted in poorly differentiated hepatocellular carcinomas with a reactivation of endogenous hTERT. These experiments demonstrate for the first time that non-cycling human mature hepatocytes can be permissive to in vitro transformation. This cellular tool provides the first comprehensive in vitro model for identifying genetic/epigenetic changes driving human hepatocarcinogenesis.
Asunto(s)
Transformación Celular Neoplásica/genética , Epigénesis Genética/genética , Transición Epitelial-Mesenquimal/genética , Hepatocitos/patología , Células Madre Neoplásicas/patología , Animales , Biomarcadores de Tumor/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Diferenciación Celular/genética , Línea Celular , Línea Celular Tumoral , Transformación Celular Neoplásica/patología , Femenino , Células HEK293 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos BALB C , Ratones DesnudosRESUMEN
Acute promyelocytic leukemia (APL) is characterized by the specific PML-RARα fusion gene resulting from translocation t(15;17) (q22;q12). Internal tandem duplication (ITD) of the FLT3 gene has been observed in approximately 35% of APLs, and large-scale studies have identified the presence of ITD as an adverse prognostic factor for acute myeloblastic leukemia (AML) patients. Aberrant expressions of surface antigens, such as CD2, CD34, and CD56, have been found in APL, but the implications of this are not well understood. We investigated the incidence of the FLT3/ITD mutation and FLT3/D835 (I836) point mutation in 25 APL patients. Incidence ratios of FLT3/ITD, D835 (I836), and both FLT3/ITD and D835 (I836) were 36%, 36% and 8%, respectively. FLT3/ITD(+) cases showed a predominance of the bcr3 isoform (P=0.008) and M3v morphology (P<0.001). We found that all FLT3/ITD(+) cases expressed CD2 (9 of 9) more frequently than that of FLT3/ITD(-) (1 of 16) (P<0.001), while only one of the CD2(+) cases (1 of 10, 10%) did not harbor FLT3/ITD, and all CD2(+)CD34(+) cases (5 of 5, 100%) harbored FLT3/ITD. In addition, quantitative polymerase chain reaction analysis showed that FLT3 mRNA was more abundantly expressed in FLT3/ITD(+) than that in FLT3/ITD(-) (P=0.025), while there was no difference between D835(I836) (+) and D835(I836)(-) with regards to aberrant surface-antigen expression, expression levels of FLT3 mRNA, M3v morphology, and the bcr3 isoform of PML-RARα mRNA. This study demonstrates that the presence of FLT3/ITD, but not D835 (I836), is closely related to aberrant CD2 expression and high expression levels of FLT3 mRNA. Our findings also suggest that FLT3/ITD as a secondary genetic event may block differentiation at the immature stage of APL.